In vivo validation of highly customized cranial Ti-6AL-4V ELI prostheses fabricated through incremental forming and superplastic forming: an ovine model study

Cranial reconstructions are essential for restoring both function and aesthetics in patients with craniofacial deformities or traumatic injuries. Titanium prostheses have gained popularity due to their biocompatibility, strength, and corrosion resistance. The use of Superplastic Forming (SPF) and Single Point Incremental Forming (SPIF) techniques to create titanium prostheses, specifically designed for cranial reconstructions was investigated in an ovine model through microtomographic and histomorphometric analyses. The results obtained from the explanted specimens revealed significant variations in bone volume, trabecular thickness, spacing, and number across different regions of interest (VOIs or ROIs). Those regions next to the center of the cranial defect exhibited the most immature bone, characterized by higher porosity, decreased trabecular thickness, and wider trabecular spacing. Dynamic histomorphometry demonstrated differences in the mineralizing surface to bone surface ratio (MS/BS) and mineral apposition rate (MAR) depending on the timing of fluorochrome administration. A layer of connective tissue separated the prosthesis and the bone tissue. Overall, the study provided validation for the use of cranial prostheses made using SPF and SPIF techniques, offering insights into the processes of bone formation and remodeling in the implanted ovine model.


Design & manufacturing of the guiding mask
The experimental investigation has been driven by the fundamental goal to customize the damage and reconstruction procedure, according to the specific needs of the implants used for humans.For this reason, a robust step by step approach was set combining geometrical, surgical and manufacturing issues.First of all, each test animal was subjected to a computerized tomography (CT) scan to obtain the virtual model of the whole skull thorough a reverse engineering reconstruction.Such a preliminary step was performed for obtaining a virtual model of the portion of the skull in which the prosthesis will have to be implanted, which is fundamental for the virtual modelling of the defect to be created.

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The skull geometry was then processed as an STL file in order to be manipulated by means of CAx tools 29 in order to define the prosthesis geometry and design its manufacturing process.
The virtual geometry reconstruction is mandatory not only for identifying the most suitable morphology of the defect and the consequent prosthetic geometry, but also for creating a customized "guide-mask" to be used by the surgeon during the phase of the "intentional" creation of the defect on the cranial case, in order to ensure the best positioning and reduce the geometrical defects due to the manual operations.Specifically, such masks were designed according to the specific anatomical conformation of the sheep; they allowed to minimize the positioning error by the surgeon, making the operations for generating the damage highly repeatable and less affected by positioning errors.Of course, each mask was set according to the hypothetical dimension of the damage, fixed as an elliptical profile, characterized by the size of the major axis and the minor axis respectively equal to 34 mm and 28 mm (the size of the defect, according to what reported in literature, could be considered critical) 30,31 .According to the surgeon's suggestions, the identification of damage was carried out pointing out 4 reference points that could be easily identified on each specimen.Figure 1 shows the 4 defined points: points 1 and 2 identify an area immediately adjacent to the point of attachment of the nuchal tendons to the skull, whereas locations 3 and 4 (6 mm away from opposite sides with respect to the segment joining the points 1 and 2) identify the sagittal plane.Figure 1 also shows the position of the defect and the mask.
The latter, although custom, also has an elliptical shape with a constant offset of 11 mm with respect to the elliptical edge of the defect.This dimension was chosen as a compromise between minimizing the invasiveness of the surgery and the need to precisely identify the "fitting" of the mask to the skull.

Design and manufacturing via SPIF and SPF
The prostheses' manufacturing chain is briefly recalled in Fig. 2 (a more detailed description of the approach is reported in 32 ): after the creation of the 3D model from the DICOM files (step "a"), the damage area was defined and located (step "b").The geometry of prosthesis (step "c") was designed by offsetting the elliptical shape geometry of the defect of a quantity equal to 4 mm.After the process design based on numerical simulation, the prosthesis was eventually manufactured (step "d").
Regarding the SPIF process, the process design passed through the 3D shape positioning of the flat sheet as well as the tool trajectory to compensate the intrinsic inhomogeneous thickness distribution.An example of one of the CAD models that reproduces the profile for obtaining the prosthesis is reported in Fig. 3.A CNC milling machine (Mazak Nexus 410), equipped with a heating chamber to increase the working temperature (up to 450 °C), was used to manufacture the implants by SPIF adopting punch diameter of 6 mm whose trajectory was calculated by a CAD/CAM software.The final geometry was isolated from the formed blank and the 4 anchoring holes subsequently drilled.The final part was characterized by a deviation from the designed CAD geometry of around 0.12 (± 0.01 mm) mm 32 .
Basically, for the SPF process the CAD geometry of the final implant (the output in Fig. 2, step "c") was used to determine the geometry of the die cavity to be copied by the blank during the forming operation (see Fig. 4).
The numerical process simulation allowed to determine the gas pressure profile able to deform the blank under an optimal strain rate level (the determination of the optimal value came from a former investigation 33 ). Figure 5a shows three gas pressure profiles (maximum value 1.5 MPa) used for the manufacturing of three different customized implants.www.nature.com/scientificreports/A universal testing machine (model 4485, produced by Instron, www.instr on.com, Norwood, MA, USA) controlled by a dedicated software (testXpert, developed by Zwick/Roell, www.zwick roell.com, Ulm, Germany) and equipped with a specifically designed equipment was used (see Fig. 5b) for the SPF tests.The set-up was heated up to 850 °C by means of an electric split furnace: to reduce the process costs, a cylindrical metal frame containing the ceramic insert with the negative geometry of the prosthesis was adopted.The Ti6Al4V-ELI blank (supplied by RL3 s.r.l., thickness 1 mm, diameter 75 mm) was deformed according to the gas pressure profile determined from the numerical simulation.The final prosthetic geometry (overall geometrical deviation of 0.02 mm with respect to the CAD geometry 32 ) was extracted by means of a wire electrical discharge cutting operation.The formed blanks, irrespective of the specific manufacturing route, were characterized by comparable surface roughness in the order of few microns, measured using the Mitutoyo Surftest SV-2000 (Mitutoyo, Kawasaki, Japan).

In vivo study in sheep model
All methods discussed in the following sections were performed in accordance with the relevant guidelines and regulations on the protection of animals used for scientific purposes (European Directive 2010/63/EU and Italian Law by Decree 26/2014).The in vivo study was conducted at the Department of Emergencies and Organ Transplantation of the University of Bari.The experimental protocol and surgical procedures were firstly approved by the Animal Welfare Body (IRB) of the University of Bari-Aldo Moro and then authorized by The Italian Ministry of Health (authorization number N° 654/2020 PR on March 5, 2019).The study has herein been reported following the ARRIVE guidelines.The sample size of four sheep for each type of prosthesis and experimental time was calculated by using the G*Power software v.3.1.9.6 (Kiel University, Kiel, Germany) and considering a power of 0.80, a type I error α = 0.05, and assuming that one prosthesis could be superior to the other in terms of bone tissue response with an effect size f greater than 0.40.

Study design
The study evaluated the performance of both SPF and SPIF prosthesis for cranial defect reconstruction in 16 healthy female Bergamasca sheep, (2 years old, 50-60 kg in weight).After selection, animals were housed in the animal facility at the department DETO of the University of Bari with six animals per fence and acclimatise for 15 days prior the surgery.They had free access to water and food, as well as exposure to natural daylight.Feeding was withheld 24 h before surgery.
The sheep were randomized into two study groups to receive either the prostheses SPF (n = 8) or SPIF (n = 8).These were further divided into two subgroups according to experimental time, the first to be euthanized after three months (Subgroup 1; n = 4) and the second after six months (Subgroup 2; n = 4).A sham negative control was not used because the procedures are not consistent with those employed in the present study and would have led to discomfort not justified.As for positive control, the material used in the study is already of proven use in clinical practice, while there is no indication on production processes that can be considered reference standards.
An incision was then made at the level of the parietal bone, and the custom-made mask was attached to the bone using 2 mm screws (Fig. 6A); the distance between the center of the holes and the inner edge of the guiding mask (corresponding to the geometry of the defect) was equal to 2 mm.Ostectomy through Piezos-urgery® (Mectron SpA, Carasco GE, Italy) was then performed (Fig. 6B) [34][35][36] .The bone gusset and mask were then removed, and the device replaced the bone gusset before having securely fastened it using the same holes (Fig. 6C).Subsequently, tissues were sutured, and the sheep were awakened from the anesthetic state.All the surgery procedures had a duration of approximately 45 min.

Euthanasia
Upon reaching the experimental times, sheep were pharmacologically euthanized with an overdose of 20-30 mg/ kg propofol, after which the heads were analyzed with CT scans to evaluate the effects of the implant on the bone.A peri-implant bone-crown of about 1-1.5 cm thick was explanted, together with the prosthesis via a craniotomy conducted by Piezosurgery® with an instrument setting of cortical bone cut using an OT 12 tip (micro-bone saw 0.35 mm with a blade having a circular cutting edge).

Bone sample preparation for microtomography and histology
Architectural characteristics and local histo-pathological response were assessed on resin-embedded specimens by micro-CT and histology, respectively.Bone segments were fixed in 4% buffered paraformaldehyde for seven days, washed under running water for 24 h, and dehydrated in a graded series of alcohol/water solutions for 48 h each (50%, 70% and two passages in 95%), followed by complete dehydration in absolute alcohol (two passages) and finally embedded in polymethylmethacrylate (Merck KGaA, Darmstadt, Germany).The specimens were sectioned along the sagittal axis and the two left and right semicircles obtained were further cut along the transversal axis with the EXAKT 310CP precision cutting system (EXAKT Apparatebau, Nordestedt, Hamburg, Germany), obtaining four quadrants: Ar (Anterior right), Al (Anterior left), Pr (Posterior right) and Pl (Posterior left), as shown in Fig. 7A The left anterior quadrant (Al) of each sample was used for microtomographic evaluations.
Consecutive sections from the medial surfaces of the Ar and Pl quadrants and from the posterior surfaces of the Al and Pr quadrants were abrasively grinded, polished and thinned to a final thickness of 50 ± 10 μm (Saphir 550, ATM GmbH, Mammelzen, Germany).The histological sections of Ar and Pl correspond to the major axis of the prosthetic implant, while those of Al and Pr correspond to the minor axis (Fig. 7B).In particular, the sections related to the major axis concern two different anatomical segments; the parietal bone (Ar) and the occipital bone (Pl) respectively.The sections relating the minor axis concern the right parietal bone (Pr) and the left parietal bone (Al) or, in some cases, the lambdoid suture.Unstained sections were used for dynamic histomorphometry and after having stained with Stevenel Blue and counterstained with Picrofucsin, according to Van Gieson method, were used for static histomorphometric measurements and qualitative histological evaluations.

Microtomography
All Al samples were scanned in the Skyscan 1172 (Bruker Micro-CT, Belgium) at a nominal resolution (pixel size) of 36 μm employing an aluminium-copper filter 1 mm thick and an applied x-ray tube voltage of 100 kV.Camera pixel binning of 4 × 4 was applied.The scan orbit was 360 degrees with a rotation step of 2 degrees.Reconstruction was carried out with a modified Feldkampii algorithm using the SkyScanTM NRecon software accelerated by GPUiii.Ring artefact reduction and beam hardening correction were applied.Volume of interest selection, segmentation to binary and morphometric analysis were all performed using SkyScan CT-Analyser ("CTAn") software.Firstly, orienting the reconstructed micro-CT images virtually allowed for the definition of a radial cutting plane that passes between the implant (i.e.defect) centre and the bisector of the inner implant angle with respect to the skull, as viewed from above (Fig. S1).On that plane, the bone tissue was divided into bone volumes of interests (VOIs), numbered in a progressive manner from the cranial site outside the implant (VOI-0) towards the centre of the defect (VOI-1 inside the implant's host bone and VOI-2 at the defect site).Figure 8A depicts the VOIs.To avoid image misreading due to metal artefact around the implant, the analysed VOIs distanced about 600 μm from the implant (Fig. 8A); consequentially, the eventual bone tissue quotes close to the prostheses were ignored in this phase, but considered in the histological one.
For morphometric analysis, the following parameters, defined by the American Society for Bone and Mineral Research (ASBMR), were measured: -BV/TV (%): percentage of bone volume defined as ratio between bone volume (BV) inside the VOI and total volume (TV) of VOI; www.nature.com/scientificreports/-Tb.Th (mm): trabecular thickness measured starting from the skeletonization of the trabecular structures, identifying their median axis and proceeding to measuring their local thickness by a "sphere-fitting" for all the voxels lying on that axis [37][38][39] ; -Tb.Sp (mm): trabecular spacing, i.e. the thickness of the spaces inside the VOI; -Tb.N (1/mm): trabecular number, i.e. the number of crossings for unit of length done by a random linear path through a trabecula inside the VOI.
In addition, a semi-quantitative evaluation of the achieved defect closure was performed.In the central micro-CT radial section, the ratio between the line segment connecting the implant's external margin and the margin of the grown bone, and the line segment parallel to the previous one and connecting the implant's internal and external margins, was defined as a percentage (Fig. S2). ).The analysis was carried out on images at a resolution of 1946 × 942 pixels on which 3 regions of interest (ROIs) were identified (Fig. 8B).The histomorphometric parameters were measured as defined by the "American Society of Bone and Mineral Research (ASBMR)" 40 :

Static and dynamic histomorphometry
-Bone Volume (BV/TV, %): percent of total volume of ROI that is occupied by bone (BV), calculated by applying the following formula: where Tb.N is the number of trabecula and is calculated as followed: -Trabecular separation (Tb.Sp, mm) calculated by applying the following formula: www.nature.com/scientificreports/-Percentage of bone defect repair (%): calculated as the percentage ratio between the distance measured from the margin of the surgical defect at the front of bone growth towards the center of the defect and that measured by the margin of the surgical defect at the center of the defect itself; -Bone-to-Implant-Contact (BIC, %): the percentage ratio between the length of bone-implant contact and the perimeter of the implant; Unstained sections were used for dynamic histomorphometry by means of the semiautomatically light/fluorescence microscope (BX51, Olympus Optical Co. Europe GmbH, Germany) connected to the XC50 Olympus digital camera (Olympus Optical).The dynamic histomorphometric analyses were performed by means of Cell*B Soft Imaging System (Olympus Imaging Solutions GmbH).The same software was used for the acquisition of the qualitative histological images.The dynamic histomorphometric paramenters were measured as defined by the "American Society of Bone and Mineral Research (ASBMR)" 40

Statistical analysis
Statistical analysis was conducted using R software v.4.2.1 (R Development Core Team, 2022) and additional R packages: 'lmerTest' v.3.1-3 41,42.Continuous data were presented as Mean ± standard error of the mean (SE) and at a significant level of p < 0.05.Before carrying out the inferential analysis of the data, their normal distribution (Shapiro-Wilk test of normality) and the homogeneity of variance (Levene test) were checked.For the microtomographic measurements, linear models were used with 'type of prosthesis' , ' experimental time' , 'VOIs analysed' as fixed factors.Therefore, for the repeated static and dynamic histomorphometric measurements obtained from the 4 quadrants of each prosthesis, linear mixed models (LMM) were used by considering 'type of prosthesis' , 'experimental time' , 'ROI analyzed' and/or 'time of administration of fluorochromes-fluorochrome time' data as fixed factors, while 'quadrants' was considered as random factor.Pairwise comparisons of the estimated marginal means were performed as a post-hoc test to identify any significant differences, adjusting p-values according Sidak's correction.
No significant difference in percentage of defect closure were observed between the SPF and SPIF prostheses between 3 and 6 months.SPF showed a mean closure of 78 ± 12% at 3 months (with 1 of 4 samples showing complete closure) and a mean closure of 95 ± 5% at 6 months (with 3 of 4 samples showing complete closure).Meanwhile, SPIF showed an average closure of 67 ± 16% at 3 months (with 1 of 4 samples showing complete closure) and an average closure of 79 ± 15% at 6 months (with 2 out of 4 showing complete closure).

Static and dynamic histomorphometry
Regarding static histomorphometric results, Fig. 10B and Fig. S3 report BV/TV (%), Tb.Th (µm), Tb.N (1/mm) and Tb.Sp (µm) measurements.No significant interactions of the selected factors ('type of prosthesis' , 'experimental time' , 'ROI analyzed') on histomorphometric parameters were found.For BV/TV, significant effects of 'type of prosthesis' factor were highlighted (BV/TV: F = 5.12, p = 0.025, resulting in significantly higher BV/TV 9%) values for SPF in comparison to SPIF.Significant differences for BV/TV, Tb.Th, Tb.N and Tb.Sp results were observed among ROI analyzed (significant effect values are reported in Fig. 10 and Fig. S3).A significant lower measurement was observed for the BV/TV (%) in ROI-2 respect to ROI-1 (BV/TV: -60%) and ROI-0 (BV/ TV: -54%).These values are typical for new bone formation process, characterized by thin trabeculae and wider inter-trabecular spaces due to their spatial distribution in the bone defect.Furthermore, these variations are independent from the type of implanted prosthesis and the experimental time.No significant differences were found between experimental times for all static histomorphometric parameters.As regard BIC (min%-max%-SPF at 3 months: 0-22; SPF at 6 months: 0-39; SPIF at 3 months: 0-3; SPIF at 6 months: 0-29) and percentage of bone defect repair (min%-max%-SPF at 3 months: 13-100; SPF at 6 months: 40-100; SPIF at 3 months: 42-100; SPIF at 6 months: 0-100) results, no significant differences were observed between the types of prosthesis and the experimental times considered.Dynamic histomorphometric analysis was carried out on unstained sections observed at ultraviolet light (UV); Supplementary Fig. S4 displays a representative image of each type of prosthesis for each experimental time.Due to differences in the experimental time in which the fluorochromes were administered to the animals, statistical analysis of the dynamic histomorphometry data was conducted separately for the data gathered at 3 and 6 months (Fig. 11).The statistical analysis revealed that there were no significant interactions between the factors that were considered, namely 'type of prosthesis' , 'ROIs analyzed' , and 'fluorochrome time' , regarding dynamic histomorphometric parameters.However, the effects of these factors are shown in the legend of Fig. 11.After 3 months, the growth of the bone mineralization surface area (MS/BS) was found to have slowed down (-18%, p = 0.041) when compared to 2 months, and the MS/BS results of the SPIF prosthesis indicated a higher value (26%, p = 0.026) than those of the SPF.At 5 months, there was an increase in MS/BS of approximately 33% (p < 0.0005) compared to 4 weeks, with a subsequent decrease of 54% (p < 0.0005) at 6 months; between 3 and  www.nature.com/scientificreports/6 months, the SPF prosthesis had higher MS/BS values (30%, p < 0.0005) compared to the SPIF.Furthermore, there was a 26% reduction in MAR values at 3 months compared to 2 months (p = 0.007), with higher mineral apposition activity in ROI-2 compared to ROI-1 (54%, p < 0.005) and ROI-0 (53%, p < 0.005).The sole noteworthy difference in MAR at 6 months was the higher rate with the SPF prosthesis compared to SPIF (30%, p < 0.0005).Finally, a 33% (p = 0.002) and 28% (p = 0.003) reduction in BFR/BS was observed at 3 months compared to 2 months and 6 months compared to 5 months, respectively.Additionally, at 6 months, the BFR/BS values of the SPIF prostheses were 57% (p = 0.002) higher than those of the SPF prostheses.

Histology
Figure 12 shows representative histological images of the SPF samples at 3 (Fig. 12A) and 6 months (Fig. 12B) and of the SPIF samples at the same experimental times (3 months: Fig. 12C; 6 months: Fig. 12D).The prostheses were well recognizable at both experimental times and in some cases one of the screws for fixing them was visible (Fig. 12D).In few sections, only the impression of the prosthesis was visible, resulting from the grinding and polishing processes of the histological specimens (Fig. 12B).Moving from the outer portion of the defect (where the prosthesis rests on the cortical bone) to the centre of it, different histological patterns were seen in correspondence of the interface between the host bone and the prosthesis.In some sections bone growth was observed in proximity mainly with the prosthetic portion corresponding to the ROI-1 region, in correspondence with the support point.No direct apposition of newly formed bone was observed in contact with the prosthesis in most of the sections: connective tissue separating the inner part of the prosthesis from the bone tissue below was present and, in some cases, it formed a fibrous capsule, which surrounded the outer edge of the prosthesis (Fig. 13A,B).A bone growth front that extends from the margin of the surgical defect (ROI-1) toward the centre of the defect, partially involving portions of ROI-2, was clearly visible.This bone growth front is evidenced by the picrofucsin staining, which highlights the newly formed bone with a more intense colour than the pre-existing one, by the more disorganized tissue architecture with thinner trabeculae and by the presence of necrotic tissue exactly localized in correspondence to the margin of the surgical defect created (Fig. 13C,D).Islets of new bone were also spotted in certain samples that lacked contact with the native bone, specifically in the ROI-2 on the dura side (Fig. S5).
As evidenced by Stevenel's Blue staining, the new bone formed in correspondence of the defect was characterized by an intense osteoblastic activity resulting in new bone matrix deposition and different mineralization grade due to bone remodeling (Fig. 13E,F).Furthermore, remodelling phenomena of the external cortical bone, in the contact zone between the prosthesis and the peri-implant host bone (Fig. 13G,H) were observed.These phenomena were characterized by the concomitant presence of areas of cuboidal osteoblast cells that lay bone matrix and areas of osteoclasts in correspondence of erosion zones (Fig. 13 I,J).

Discussion
Two highly customized prostheses, manufactured using SPF and SPIF techniques, were evaluated microtomographically and histologically for their effect on bone tissue in an in vivo craniectomy sheep model.The study examined inflammatory reactions, new bone deposition, and repair of bone defects.The manufacturing process used to create titanium prostheses was previously demonstrated in other studies 1,23 .Briefly, the conversion of the DICOM images to CAD models improves the optimal definition of the implant's geometry.SPF implants, where found to be more geometrically accurate (with an average deviation from CAD geometry of only 0.02 mm) when compared to those manufactured by SPIF.However, they required a longer manufacturing time due to the need for pre-heating of the tools and a longer forming step.Furthermore, the creation of the guiding mask to ensure accurate implant positioning during surgery, minimized the risk of micro-motion and mismatch issues with the surrounding peri-implant bone.These adopted strategies were able to guarantee the most suitable prosthesis fit to sheep skull anatomy 1 .
No significant inflammatory response or side effects were observed in the implant site.Additionally, the healing of soft tissue was normal and aligned with expected scarring times.The healing processes of craniomaxillofacial bone defects have been extensively documented in the literature across various animal species 43 .Ratsand rabbits are the most commonly used preclinical animal models, but sheep have bone dimensions similar to those in humans, along with comparable turnover and bone modeling rates 44 .When the defect size exceeds 22 mm in diameter, it is considered a critical size defect in sheep 45 .Hubbe et al. replicated this defect in an ovine model's skull and covered it with a 3D-printed porous titanium-based scaffold.The scaffold was either coated with hydroxyapatite (HA) or left uncoated, and filled with or without bone cement.Histological analyses showed that bone ingrowth was significantly higher in the HA-coated group, starting from the lateral side of the defect, and extending up to the scaffold center 30 .
According to the current microtomographic and histomorphometric results, bone formation was found to start from the native bone, although full-thickness closure of the cranial theca was not observed in any of the sixteen animals.Omar et al., carried out the regeneration of large ovine cranial defects using 3D-printed cranial implants consisting of a titanium-reinforced bioceramic (BioCer) in comparison to titanium control materials 46 .After 12 months, titanium implants exhibited less bone restoration and BIC values (center, 3%; periphery, 19%) compared to BioCer implants which promoted a higher degree of bone formation, remodeling and osseointegration.However, they observed the same bone formation mode reported in this study that primarily occurred along the existing borders of the bone, with a lesser extent in the defect center.As well as, Gallinetti et al. 47 studied the repair of cranial defects in sheep using a titanium mesh either uncoated or coated with calcium phosphate (CaP).Significantly higher bone neoformation (p < 0.001) with moderate osteoconduction (p < 0.001) and a marked osteointegration (p < 0.001) were observed in the CaP-Ti cover versus the Ti-mesh.They also observed that bone formation started from the native bone and complete closure did not occur in any animal.Additionally, they confirmed that bone growth mainly occurred on the dura side rather than in close proximity to the implants, which were observed on the skin side.This aligns with our histological discovery of new bone growth spots near the dura side but not in direct contact with the native bone.This is likely due to the presence of skeletal stem cells (SSCs) in the periosteal layer of the dura mater, which play a key role in bone regeneration 48,49 .The osteopromoting effects of the dura mater are also reported in the study of Piitulainen et al., where a large calvarial bone defect of a child was reconstructed with a bioactive and biostable nonmetallic implant.New bone was only found on the inner surface of the implant, suggesting the importance of chemical and biological engagement of the dura mater in the regeneration of the bone defect 50 .Despite the SPF and SPIF implants in this study mimicking the shape of the cranial vault and possessing adequate mechanical properties, they differ from the aforementioned studies in that they do not completely fill the entire defect thickness, lack a porous structure to promote vascularization and bone ingrowth and are not reinforced with osteoinductive and absorbable material such as CaP.Furthermore, they have a smooth surface that does not promote cell adhesion.The cranial defects repaired without direct contact with the prosthetic devices.This is shown by Figs. 8 and  12, where a narrow space is easily detectable between the re-grown bone and the inner surface of the Ti implant.Qualitative analysis the histological images concluded that the newly generated bone followed the curvature of the brain rather than the shape of the Ti implant.In particular, no osteoconductivity properties were observed near the implants, which were still separated by a thin layer of fibrous tissue.Connective tissue growth underneath the implants could be caused by various factors, including inadequate peripheral healing (i.e.anchoring point) between the material and the bone, resulting in connective tissue ingrowth through the external margins.These results suggest that SPF and SPIF implants do not directly participate and stimulate bone regeneration in the cranial defect.Instead, they provide necessary protection and rigidity, acting as a barrier membrane in guided bone regeneration (GBR), thus sustaining the space of the regenerating bone area beneath it [51][52][53] .Due to its excellent mechanical properties, titanium frameworks are commonly used in the dental GBR technique, mostly embedded inside polytetrafluoroethylene (PTFE) membranes in order to provide additional stability and adaptation to the bone defect 54 .The decision to employ titanium in GBR drew inspiration from the positive results achieved when utilizing a titanium mesh to reconstruct maxillofacial defects [55][56][57][58][59] .Finally, bone resorption observed on the external surface of the cranial theca, specifically where the prosthesis is implanted on native bone, it may occur due to physiological remodelling activity of the cortical bone in response to mechanical stresses, such as stress-shielding, experienced by the fixation screws.
A possible limitation of the current study could be the lack of control implants, but to our knowledge the available options for cranioplasty, apart from autologous bone grafts, are typically titanium-based (like our devices) or made from acrylic materials 60 .The analysis of the literature, with particular reference to clinical studies, supports that there is no standard production process for cranial prosthesis, due to the use of diverse surgical approaches, materials, and manufacturing methods.Studies are predominantly oriented towards the use of methods that allow the creation of customized devices, based on preoperative CT imaging, overcoming the use of prefabricated prostheses to be shaped in a surgical procedure.Even comparing various titanium implant production methods does not yield a clear universal approach for both customized and non-customized prostheses.This entails an ethical and scientific difficulty in selecting a reliable and recognizable control.Therefore, the design of the present pilot study, relying on the use of device produced using a material with proven biocompatibility and orthopaedic effectiveness such as titanium, did not foresee a further group with prefabricated prostheses [61][62][63][64] .Furthermore, according to the principles of the 3 R's (Refinement, Reduction, Replacement) and in our opinion, the inclusion of a control material group would have had a further impact on the total number of animals used rather than the results of the study.Similarly, not comparing the results with a sham defect could be considered a limitation.Our decision to omit such control defects, since the sham technique involves craniotomy, removal of the bone segment and its immediate repositioning and fixation to the skull.In addition, the method of attachment of the autogenous graft to the skull would have been different from that used in the prostheses tested, which would have reduced the ability to withstand external mechanical stresses of a pressor type.Such stresses could be caused by social communication behaviour or scratching and itching of the sheep, with the risk of damage to brain structures.Further investigations could be implemented in future studies to provide a more detailed and in-depth characterization of the quality and state of the regenerated bone tissue.Techniques such as Raman spectroscopy or FTIR could be used for this purpose.

Conclusions
In this study, a novel approach combining mechanical and surgical techniques was proposed.The adoption of two innovative and flexible sheet metal forming processes -the SPF and the SPIF allowed to achieve the geometrical complexity necessary to ensure a high level of correspondence between the formed components and the bone's region surrounding the defect.
The conducted in vivo test highlights the absence of any adverse effects or inflammatory reactions, indicating strong mechanical fixation of the prosthesis to the adjacent bones, serving as evidence for the high precision achieved through this methodology.The implants under study offer indirect support for bone regeneration by providing protection and rigidity.Bone filling involves from 30 to 40% of the defect and primarily follows the natural curve of the cranium in relation to the covering of the brain.

Figure 1 .
Figure 1.The 3D overview of the guiding mask positioning on the skull along with the reference nuchal tendons' attachment points.

Figure 2 .
Figure 2. Overview of the design and manufacturing chain for the customized implants.

Figure 3 .
Figure 3. (a) 3D view and (b) lateral views of the designed geometry for SPIF process.

Figure 4 .
Figure 4. Modelled parts in the FE model of the SPF process: (a) the die cavity, (b) the Ti blank.

Figure 5 .
Figure 5. Implant manufacturing by SPF: (a) example of the gas pressure profile calculated from the FE simulation of the SPF process, (b) experimental equipment.

Figure 6 .
Figure 6.Three distinct moments of different surgeries procedure: (A) Guiding mask fixed to the bone with screws; (B) Bone gusset removal through Piezosurgery osteotomy; (C) Device implantation and fixation with screws.
Static histomorphometric measurements were performed by Fiji software, an open-source image processing package based on ImageJ (Schindelin et al. 2012, Schneider et al. 2012

Figure 7 .
Figure 7. (A) Example of specimen embedded in polymethylmethacrylate.The sagittal cutting axis and the transversal cutting axis are highlighted by the red and the yellow dashed lines respectively; (B) Representative histological images of Ar and Pl representing the major axis of the prosthetic implant and of Al and Pr corresponding to the minor axis.Stain: Stevenel Blue counterstain with Picrofucsin according to Van Gieson; scale bar: 3 mm.

Figure 8 .
Figure 8.(A) left, radial section of the microtomographic reconstruction, highlighting the areas corresponding to the volumes of interest (VOIs): VOI-0 (host bone outside the implant) surrounds the cranial site close to the implant; VOI-1 (host bone inside the implant) is 4 mm wide, i.e. more than half the difference between the implant and defect ellipsoidal axes, starting from the outer edge of the implant and extending towards the centre; VOI-2 (within the defect) starts from the end of VOI-1 and surrounds the regenerated bone.To avoid the influence of metal artefacts, the VOIs were spaced approximately 600 μm from the implant, concentrating on the bone tissue but neglecting any quotes close to the prosthesis, for which reference is made to histology.Again because of the metal artefact, but also because of the difficulty in following the actual host bone margin, the inner margin of VOI-0 and the lateral margins of VOI-1 are drawn geometrically vertical, unlike ROI-0 and ROI-1 in the histology images.On the right, 3D views of a microtomographic reconstruction (obtained with CTVox v.3.3.1 software) with the implant in light grey and the cranial bone in different colours depending on the VOI.(B), ROIs definition.ROI-0 (yellow rectangle): cranial bone immediately nearby the implant site; ROI-1 (blue rectangle): portion of the cranial bone below the prosthesis extending from the edge of the prosthesis to the edge of the bone defect; ROI-2 (green rectangle): region corresponding to the bone defect following cranial bone curvature and based on the position of the dura and prosthesis.Scale bar: 4 mm.
: -Mineralizing Surface per Bone Surface (MS/BS, %) calculated by the following formula: where sL.Pm/B.Pm is the length of the single fluorescent band of a fluorochrome expressed in relation to the length of the bone trabecula and dL.Pm/B.Pm is the length of the double fluorescent band.-Mineral Apposition Rate (MAR μm/day): measured as the distance between the midpoints of two consecutive deposited fronts of fluorochrome divided by the time between the labelling period.It represents the average speed at which individual lines of osteoid are mineralized.-Bone Formation Rate (BFR/BS μm 3 /μm 2 /day): it expresses the measurement of the amount of mineralized bone formed, per unit of bone surface per day.It is calculated adopting the following formula:

Figure 9 .
Figure 9. (A) 3D rendering of the microtomographic reconstruction of a sample with SPF prosthesis at 6 months.(B), 3D rendering of the microtomographic reconstruction of a sample with SPIF prosthesis at 6 months.There are visibly different trabeculae thickness and orientation in VOI-0 (in yellow) for these specific SPF and SPIF examples.That is, the structure of the host bone can have affected the different bone regrowth/remodelling results in respective VOI-1 (in blue) and VOI-2 (in green).However, on average, VOI-0 microtomography morphological parameters did not show statistical differences between SPF and SPIF (see Fig.10Aand Fig.S3).